The human eye is an organ of the body which is not reproducible. It is vulnerable to eye disorders such as degenerative diseases, glaucoma, or cataract in retina, cornea, conjunctiva or uvea. In particular, the cornea is a part of the outer membrane of the eyeball exposed to the outside. The cornea plays important roles in protecting the eyeball and transmitting light to the retina by refracting wavelengths entering into the eye. However, the cornea is vulnerable to injuries such as scratch or impact and so on since it does not have other protective structures except the eyelid. In addition, in case of serious damage to the cornea, there are no practical treatment options except transplantation. Unfortunately, however, it has been reported that the transplanted cornea exhibits poor biological adaptation and adverse events.
Recently, as more people are wearing contact lenses for vision correction or fashion, the number of people suffering from eye disorders such as corneal surface injury and dry eye syndrome, etc. is increasing. Since the cornea is overlaid with a contact lens, oxygen supply to the cornea is blocked and the dryness of the cornea gets worse by blocking the tear secreted at the lacrimal gland. As such, most of people wearing contact lenses frequently use artificial tears etc., to relieve the dryness of the cornea. If the dryness of the cornea get worse, the treatment of damaged cornea is essential for preventing progression to other eye disorders that can be triggered from it. Examples of treatments of corneal injury include direct application of ophthalmic drugs such as a liquid formulation, a gel or an ointment to the cornea.
Various forms of ophthalmic preparations are required to have an excellent pharmacological activity and maintain the activity through stable storage. The stability and pharmacological activity of the ophthalmic preparation are affected by its ingredient and content and the treatment conditions during manufacturing and distribution processes. Especially, in the whole manufacturing and distribution processes, the dehumidification and oxidation prevention of the ophthalmic preparation are very important. For this purpose, several methods to maintain the product quality are utilized including, for example, sealing packaging after sterilization; coloring a glass vial to prevent direct exposure to sunlight; and adding a dehumidifying agent such as a silica gel. However, even with the above various methods, it is still difficult to achieve quality maintenance depending on a seasonal temperature difference, a humidity change or oxidation. Thus, a need for an ophthalmic preparation having a good stability to maintain its pharmacological activity at a constant level is increasing.
Meanwhile, thymosin β4 is a protein discovered in the thymus in 1981, and consists of 41 to 43 amino acids, whose isoelectric point is 5.1. It was identified as an actin-sequestering molecule in animal cells by Riva in 1991, and then found to be involved in the immune regulation and neuroendocrine system. Moreover, it was found that thymosin β4 not only functions as a terminal deoxynucleotide transferase in thymocytes, but increases the migration of macrophages and its antigen and the secretion of luteinizing hormone by hypothalamic explants. It is also known to remove the toxicity of cytosine arabinoside and inhibit the cell cycle of hematopoietic stem cells to increase the adhesion and migration of endothelium.
Therefore, studies on the usage of thymosin β4 having the pharmacological activities as mentioned above for the treatment of ocular disorders were carried out. For example, Korean Laid-Open Patent Publication No. 10-2008-0033939 discloses a pharmaceutical composition to treat eye inflammations, eye infections (bacterial, fungal or viral), and glaucoma as uses of an ophthalmic solution comprising thymosin β4 and a preservative having sterilizing action. However, such pharmaceutical composition has the disadvantage that thymosin β4 that is prepared by a freeze-drying method and is labile to temperature change can be denatured depending on temperature change, which leads to the reduction of its pharmacological activity.
Korean Laid-Open Patent Publication No. 10-2015-0080026 discloses a preparation method comprising: adding a pH buffer and an antioxidant to an aqueous solution containing tetrahydrobiopterin (BH4) for oral administration, intravenous injection or co-administration with food; spraying a non-oxidizing gas thereto; and filling the aqueous solution into a container. However, since the aqueous solution containing BH4 as above is filled into a container in the presence of a non-oxidizing gas after it is prepared into a mixed solution containing BH4, a pH buffer, and an antioxidant etc. while being exposed to oxygen under the atmosphere before the filling process into container, it has the disadvantage that oxidation, addition of impurities and so on cannot be blocked during the above mixing step.
As such, the present inventors have endeavored to improve the stability of an ophthalmic preparation comprising thymosin β4 by blocking its modification, and to maintain its pharmacological activity at a constant level for long-term period, and found that stability of thymosin β4 and its ophthalmic preparation can be improved by blocking contact with oxygen using an inert gas during manufacturing and filling and sealing processes of the ophthalmic preparation.